The Role and Molecules Mechanism of Klotho in Renal Injury in Salt-sensitive Hypertension

Subjects: Medicine, Pharmacy >> Clinical Medicine submitted time 2023-03-01 Cooperative journals: 《中国全科医学》

ZHAO Wei TANG Roengji YANG Shanshan YANG Fang SUN Feng LIAN Qiufang

Abstract： Background Klotho is closely related to the occurrence and development of kidney disease. Salt-sensitive hypertension（SSH）is often accompanied by kidney disease. At present，there are few reports on the role and molecules mechanism of klotho in renal injury in SSH. Objective To investigate the role and molecules mechanism of klotho in renal injury in SSH. Methods The rat glomerular mesangial cell line HBZY1 was selected as the experimental cells on June 15，2021， and the experimental cells were divided into the control group and the model group. The model of HBZY1 cell injury induced by NaCl 137 mmol/L and angiotensin Ⅱ（Ang Ⅱ）10-6 mmol/L was used to simulate the renal injury in SSH，and the cells were collected. The differences in the expression of klotho mRNA and protein were detected by real-time fluorescent quantitative PCR （qRT-PCR）and Western Blot. The interference vector and overexpression vector of klotho and the overexpression vector of angiotensin Ⅱ type 1 receptor（AT1R）were constructed. The klotho interference experiments were divided into five groups， including the control group，empty group，klotho-siRNA1 group，klotho-siRNA2 group and klotho-siRNA3 group； The klotho overexpression experiments were divided into three groups，including the control group，empty group and klotho overexpression group；The AT1R overexpression experiments were divided into three groups，including the control group，empty group and AT1R overexpression group. The constructed vectors were transfected into cells with verified transfection efficiency. After successful transfection，the experiment was divided into two parts. The first part of the experiment was to verify the renal protective effect of klotho，the experiment subjects were divided into four groups，including the control group，model group， klotho overexpression group and klotho interference group. The second part of the experiment was to explore whether the renal protective effect of klotho was related to AT1R，the experiment subjects were divided into three groups，including the model
group，klotho overexpression group and klotho+AT1R overexpression group. After successful transfection，the tests including cell viability detected by cell counting kit-8（CCK-8） method，reactive oxygen species（ROS）content detected by flow cytometry，malondialdehyde（MDA）and superoxide dismutase（SOD）content in cell supernatant detected by enzyme-linked immunosorbent assay（ELISA），interaction effect between kltho and AT1R detected by co-immunoprecipitation（Co-IP）. Results Compared with the control group，mRNA level and protein expression of klotho in the model group decreased in model group（ t=7.102，7.506； P=0.002，0.002），klotho-siRNA2 interference effect was more significant（ P<0.001）， the expression of klotho protein in the klotho overexpression group increased significantly（ P<0.001），the expression of AT1R protein in the overexpression group increased significantly（ P<0.001）. Effects of klotho on cell viability and oxidative stress injury：Compared with the control group，cell viability in the model group decreased（ P<0.001），intracellular ROS and MDA content increased（ P<0.001， P=0.004），and SOD content decreased（ P=0.041）；Compared with the model group，cell viability in the klotho overexpression group increased（ P<0.001），intracellular ROS and MDA content decreased and SOD content increased（ P<0.001， P=0.003， P=0.018）；Compared with the model group，cell viability in the klotho interference group decreased（ P<0.001），while intracellular ROS and MDA content increased and SOD content decreased（ P<0.001， P=0.002， P=0.001）. Effects of klotho on cell viability and oxidative stress injury through AT1R：Compared with the model group，cell viability increased（ P<0.001），intracellular ROS and MDA content decreased and SOD content increased（ P<0.001， P=0.024， P=0.007）in the klotho overexpression group；Compared with the klotho overexpression group，cell viability decreased（ P<0.001），ROS and MDA content increased and SOD content decreased（ P<0.001， P=0.001， P=0.002）in the klotho+AT1R overexpression group. Co-IP determined that there was an interaction between klotho and AT1R. Conclusion Klotho plays a protective role in renal injury in SSH by inhibiting oxidative stress injury through interaction with AT1R.

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The role and molecular mechanism of Klotho in salt-sensitive hypertensive renal injury

Subjects: Medicine, Pharmacy >> Clinical Medicine submitted time 2023-01-28 Cooperative journals: 《中国全科医学》

ZHAO Wei TANG Rongjie YANG Shanshan YANG Fang SUN Feng LIAN Qiufang

Abstract：

Objective To investigate the role of Klotho in salt-sensitive hypertensive renal injury and its molecular mechanism. Methods HBZY1 cell injury model induced by NaCl 137 mmol/L and AngII 10-6 mmol/L was used to simulate salt-sensitive hypertensive kidney injury, and cells were collected. Real-time quantitative PCR (qRT-PCR) and Western Blot were used to detect the expression of Klotho mRNA and protein. The Klotho interference vector and overexpression vector were constructed, and the Angiotensinogen II receptor-1(AT1R) overexpression vector was transfected into cells. The transfection efficiency was verified by qRT-PCR and Western Blot. The following assays were performed after transfection. Cell proliferation activity was measured by CCK-8 assay, and ROS content was measured by Flow cytometry assay, the contents of malondialdehyde (MDA) and Superoxide dismutase superoxide dismutase (SOD) in the supernatant were detected by enzyme-linked immunosorbent assay (ELISA) , and the interaction between Klotho and AT1R was detected by co-immunoprecipitation (Co-IP) . Results Compared with the control group, the expression of Klotho mRNA and protein decreased in the model group (P<0.05). The KLOTHO-SIRNA2 interference effect was the best, and the expression of Klotho in the Klotho overexpression group and AT1R in the AT1R overexpression group increased, indicating the overexpression effect. Compared with the control group, the cell viability of the model group was decreased, the contents of ROS and MDA were increased, and the contents of SOD were decreased (P<0.05), the content of SOD decreased (P<0.05) , but the activity of klotho-treated cells decreased further, the content of ROS and MDA increased further, and the content of SOD decreased further (P<0.05). Compared with the model group, the Klotho overexpression group had higher cell viability, lower ROS and MDA contents and lower SOD contents (P<0.05) , while the Klotho + AT1R overexpression group had lower cell viability, higher ROS and MDA contents, the content of SOD decreased (P<0.05). CO-IP determines the interaction between Klotho and AT1R. Conclusion Klotho plays a protective role in salt-sensitive hypertensive renal injury by inhibiting oxidative stress injury through interaction with AT1R.

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